The overall objective of Project 1 is the design and construction of novel vaccines against SIV and HIV-1 and their evaluation in immunization strategies aimed at eliciting broadly reactive humoral and cellular immunity against primate lentivirus infection and disease. Because the limits of vaccine protection have yet to be defined, it is very important to expand upon successful vaccination and challenge systems such as SIVmne challenge and to determine the limits of protection. The proposal is based on the substantial progress made during the last NCVDG and draws on the expertise and experience of the co-project leaders developed over the last five years at the University of Washington/Bristol-Myers Squibb and the University of California/Chiron NCVDG groups. Specific Aim 1 is designed to expand the already extensive collections of recombinant vaccinia virus and stable cell lines to express the envelop antigens (gp120/130 and gp160), the core antigens, or the pseudovirions of two SIC (SIVmne and SIVsmm) and two HIV-1 (IIIB and MN) virus isolates. These immunogens will be used alone or in combination with live recombinant virus priming for the immunization of macaques. First round experiments will be done in the SIV/macaque model, and successful approaches will be tested with the analogous immunogen(s) in the HIV-1/macaque model. In Specific Aim 3, we plan to use the SIV/macaque model, and potentially the HIV-1 macaque model, to evaluate the limits of the protective immunity elicited by the "prime and boost" regimen. The role of live virus priming and the contributions of each major structural antigen in protective immunity will be studied, as outlined in Project 3. In addition, the immune responses and the protective efficacy of the "prime and boost" regimen will be compared with that of live attenuated virus vaccines. We will also vary the components of the "prime and boost" regimen to explore the effects of novel immunization approaches on the immune responses generated. This will include the use of adeno- associated virus for priming and potentially for vivo boosting (Specific Aim 3) and novel antigen presentation and formulation (Specific Aim 4). Finally, we propose to study the mechanisms of vaccine success or failure by analyzing the fine specificity of the humoral immune responses generated (Specific Aim 5, in collaboration with the SIV and HIV-1 Virology Cores), as well as the sequence variation and the genotypes of virus that breakthrough the pre-existing immunity generated by the various vaccination approaches (Specific Aim 6).